Broad challenge area (14), Stem Cells. Specific challenge topic 14-MH-101: Developing iPS cells for mental disorders. Dysfunction of GABAergic interneurons of the cerebral cortex has been implicated in a variety of major neuropsychiatric illnesses, including schizophrenia, autism, anxiety, and epilepsy (where depression is a major source of morbidity). However, our lack of knowledge of how human interneurons develop and function, and how disease-related genes influence this process, greatly hinder our ability to understand, prevent or to treat interneuron-related mental illness. In addition, since neuropsychiatric disorders such as schizophrenia and autism appear to largely result from the combinatorial effects of polygenic risk factors with a significant component of environmental influence (perhaps mainly in utero), techniques for addressing this complicated intermix of effects would be tremendously useful. Induced pluripotent stem cells (iPSCs) are an important potential source of human interneurons that could be used to address both genetic influences on interneuron development and function, and to study genetic-environmental interactions in this process. Two specific examples of ways in which an iPSC based method could be used to study schizophrenia are provided in the Future Directions section below, but the ideal future objective would be to;1) derive iPSCs from patients known to harbor particular risk alleles for the given neuropsychiatric illness together with unaffected and non-risk allele carrying controls, 2) insert a fluorescent reporter construct known to express at relevant stages of interneuron development (i.e. genesis, migration, elaboration of processes and connectivity, activity dependent refinement, mature function, senescence) that are implicated in how that risk allele affects the development of disease, 3) direct the differentiation of the iPSCs to the particular stage of interneuron development, and 4) collect cells at that stage by FACS for the study of disease-related gene and protein expression, neuronal development or function, susceptibility to models of environmental insults, and testing of preventative or corrective agents. As a first step towards this idealized objective, the goal of this proposal is to develop and validate methods of generating, isolating, and studying putative cortical interneurons from human fibroblast derived iPSCs. The proposal builds on the progress in the Anderson and Studer labs at i) using modified bacterial artificial chromosomes (BACs) to obtain tissue-specific expression of reporter genes in transgenic mice as well as in mouse and human embryonic stem cells ii) developing highly efficient protocols for the conversion of human ES cells and human iPSCs into neural cells with forebrain identities, We propose the following Aims: Specific Aim 1. Use of an Lhx6-GFP reporter to isolate iPS cells directed to interneuron lineages Specific Aim 2. Use of an Nkx2.1-GFP reporter to isolate iPS cells directed to interneuron lineages Nkx2.1 is expressed in mitotic interneuron progenitors, whereas Lhx6 is expressed by the some of the same progenitors at the time of cell cycle exit and maintained during subsequent development. By directing iPSCs to putative interneuron progenitor fates (Nkx2.1+), we can prospectively isolate cells at this critical stage for genetic and transplantation studies aimed at defining their fate potential. These studies will be complemented by the isolating cells at Lhx6-expressing stages, when most of them have down-regulated Nkx2.1. We can again perform genetic and fate potential studies particular to these later stages of interneuron development. Despite the tremendous potential for using iPS-based approaches to study mental illness, several key challenges must be overcome to realize this promise. Among the key questions are: First, what is the optimal method for deriving interneuron-progenitor like cells from iPSCs? Second, how do we differentiate them into distinct interneuron types, and what system can we use to define those types outside of the human brain? Third, how much variability is present in this method when multiple interneuron-differentiated samples derived from the same human source are compared? These questions must be addressed to achieve significant utility in the use of iPSCs to study interneuron-related disease. High variability within multiple samples from the same source will render comparisons between patient and control-derived samples meaningless, or even misleading. To address these challenges we have assembled a team with considerable experience at each level of the project;1) embryonic and iPS-derived stem cell biology (Co-PI Dr. Studer), 2) cortical interneuron development (PI-Dr. Anderson), 3) electrophysiology in forebrain slices (Co-Investigator Dr. Goldstein), and 4) human cerebral cortex/interneuron and pathobiology of schizophrenia (Consultant David Lewis). Success in achieving the goals of the project will enable a wealth of future studies by our and many other groups, directed at the interaction of disease-related genes and environment on interneuron genesis, maturation, and function. Such iPSC-based approaches could have a major impact on understanding the tremendously complex, and difficult to study, role of interneurons in neuropsychiatric diseases. PUBLIC HEALTH RELEVANCE: Dysfunction of GABAergic interneurons of the cerebral cortex has been implicated in a variety of major neuropsychiatric illnesses, including schizophrenia, autism, anxiety, and epilepsy (where depression is a major source of suffering). However, both our general lack of knowledge regarding how human interneurons develop and function, and our specific lack of knowledge of how disease-related genes may influence this process, greatly hinder our ability to understand, prevent or to treat interneuron-related mental illness. Induced pluripotent stem cells (iPSCs) are an important potential source of human interneurons that could be used to address both genetic influences on interneuron development and function, and to study genetic-environmental interactions in this process. The goal of this proposal is to develop methods and protocols for the consistent derivation of function inhibitory interneurons from human iPSCs, to lay critical ground word for future studies on the role of inhibitory interneurons in neuropsychiatric disease.